Design, preparation and application of novel IL-2, INF alpha and Fc fusion protein
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A technology of IL-2 and fusion protein, applied in the field of biomedicine, can solve problems such as weak specificity, large side effects, and short half-life
Pending Publication Date: 2022-08-05
NANJING JSIAMA BIOPHARMACEUTICALS LTD
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Problems solved by technology
[0005] In order to solve the above problems, the present invention discloses the design, preparation and application of a novel fusion protein of IL-2, INFα and Fc. Through gene mutation, the binding ability of IL-2 and its receptor, INFα and its receptor can be changed to solve the current problem. It has the disadvantages of weak drug specificity, short half-life and large side effects
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Embodiment 1
[0059] ELISA detection and receptor IL-2Rα protein binding ability
[0060]The packages are different (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.0625ng / ml, 9.765625ng / ml, 2.44140625ng / ml, 0). Laoshenzhou), 100UL / holes at 4 ° C; closed with 3 % skim milk powder at 37 ° C for 1h; add 1ug / ml fusion protein per hole and 100ul each of its control samples, and incubate 1h at 37 ° C; then add HRP -labeled sheep anti -human IL IL IL people IL. -2PAB, incubation at 37 ° C for 1h, after 10 minutes of color rendering, read OD450 on the enzyme label. Consequences figure 2 Essence
Embodiment 2
[0062] ELISA detection and receptor IL-2Rβ protein binding ability
[0063] The packages are different (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.0625ng / ml, 9.765625ng / ml, 2.44140625ng / ml, 0). Laoshenzhou), 100UL / holes at 4 ° C; closed with 3 % skim milk powder at 37 ° C for 1h; add 1ug / ml fusion protein per hole and 100ul each of its control samples, and incubate 1h at 37 ° C; then add HRP -labeled sheep anti -human IL IL IL people IL. -2PAB, incubation at 37 ° C for 1h, after 10 minutes of color rendering, read OD450 on the enzyme label. Consequences image 3 Essence
Embodiment 3
[0065] ELISA detection and receptor IFNαr2 protein binding ability
[0066] The packages are different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.0625ng / ml, 9.765625ng / ml, 2.44140625ng / ml, 0) protein (10359-H02H, Yiliang Shenzhou ), 100UL / hole at 4 ° C overnight; closed 3 % skimmed milk powder at 37 ° C for 1h; add 1ug / ml fusion protein per hole and 100ul each of its control samples, incubate 1h 37 ° C; then add rabbit anti -human interferon 2,37 ℃ Incubation 1h, then add HRP -labeled sheep anti -rabbit H+L, incubate for 1h 37 ° C, and after 10 minutes of color rendering, read OD450 on the enzyme label. Consequences Figure 4 Essence
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Abstract
According to the invention, modifications aiming at human interleukin-2 (IL-2) and interferon alpha (IFN alpha) are included; the fusion protein is formed by IL-2, INF alpha or IL-2 / INF alpha double factors and Fc after modification; as well as the design, preparation and use of these fusion proteins; respectively fusing the mutant IL-2 and the mutant INF alpha (double factors) at the N terminal or the C terminal of the Fc; fusing the mutant IL-2 at the N terminal or the C terminal of the Fc; respectively fusing the mutant INF alpha at the N end or the C end of the Fc; l234A / L235A mutation is introduced into the Fc segment at the same time, so that the binding capacity of the Fc segment and a receptor FcgRIII is reduced; m252Y / S254T / T256E mutation is introduced, so that the half-life period of the protein is prolonged; through gene mutation, the binding capacity of IL-2 and a receptor thereof and the binding capacity of INFalpha and a receptor thereof are changed, mutants better than wild type mutants are obtained, the binding capacity with NK cells and CD8 + T cell receptors is improved, and the defects that existing drugs are weak in specificity and large in side effect are overcome.
Description
Technical field [0001] The invention is a biomedical technology field, which involves the design, preparation and use of the new IL-2 and INFα and FC fusion proteins. Background technique [0002] IL-2, also known as T cell growth factor, is the gene is located in No. 4 chromosomes, including a sequence of 7KB, consisting of 133 amino acids, and the molecular weight is about 15kd. IL-2 acts through IL-2R. IL-2R includes three subunit, IL-2Rα (ie CD25), IL-2Rβ (ie CD122), and IL-2R γ (ie CD132). Three sub-submissions can form three forms of receptor: high binding force receptor contains all three sub-meter IL-2Rα / β / β / β, and the binding force receptor contains IL-2Rβ / γ two sub-macro Receptor IL-2Rα. Among them, IL-2Rβ and IL-2R γ are necessary for IL-2 to activate the downstream signal pathway. When IL-2 is combined with IL-2Rβ and IL-2Rγ at the same time, the two receptor subunit forms a heterogeneous diode, phosphorylationization The STAT5 in the cells enters the corresponding ge...
Claims
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